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•The reliance on anaerobic metabolism causes fatigue during high-intensity exercise.•Metabolic pathways maintain ATP at levels that do not impair contractile function.•Intracellular homeostasis is disrupted by metabolite accumulation, namely H+and Pi.•Multifaceted and synergistic effects of H+and Pimarkedly impair muscle contraction.•Fatigue has a bioenergetic basis determined by the rate and extent of metabolite accumulation.

Nemaline myopathies (NMs) are a group of congenital muscle diseases caused by mutations in at least 10 genes and associated with a range of clinical symptoms. NM is defined on muscle biopsy by the presence of cytoplasmic rod-like structures (nemaline rods) composed of cytoskeletal material. Myofiber smallness is also found in many cases of NM and may represent a cause of weakness that can be counteracted by treatment. We have used i.p. injection of activin type IIB receptor (ActRIIB)–mFc (an inhibitor of myostatin signaling) to promote hypertrophy and increase strength in our prior murine work; we therefore tested whether ActRIIB-mFc could improve weakness in NM mice through myofiber hypertrophy. We report a study of ActRIIB-mFc treatment in the Acta1H40Y mouse model of NM. Treatment of Acta1H40Y mice produced significant increases in body mass, muscle mass, quadriceps myofiber size, and survival, but other measurements of strength (forelimb grip strength, ex vivomeasurements of contractile function) did not improve. Our studies also identified that the complications of urethral obstruction are associated with mortality in male hemizygote Acta1H40Y mice. The incidence of urethral obstruction and histologic evidence of chronic obstruction (inflammation) were significantly lower in Acta1H40Y mice that had been treated with ActRIIB-mFc. ActRIIB-mFc treatment produces a mild benefit to the disease phenotype in Acta1H40Y mice.

Slow type I fibers in soleus and fast white (IIa/IIx, IIx), fast red (IIa), and slow red (I) fibers in gastrocnemius were examined electron microscopically and physiologically from pre- and postflight biopsies of four astronauts from the 17-day, Life and Microgravity Sciences Spacelab Shuttle Transport System-78 mission. At 2.5-μm sarcomere length, thick filament density is ∼1,012 filaments/μm2in all fiber types and unchanged by spaceflight. In preflight aldehyde-fixed biopsies, gastrocnemius fibers possess higher percentages (∼23%) of short thin filaments than soleus (9%). In type I fibers, spaceflight increases short, thin filament content from 9 to 24% in soleus and from 26 to 31% in gastrocnemius. Thick and thin filament spacing is wider at short sarcomere lengths. The Z-band lattice is also expanded, except for soleus type I fibers with presumably stiffer Z bands. Thin filament packing density correlates directly with specific tension for gastrocnemius fibers but not soleus. Thin filament density is inversely related to shortening velocity in all fibers. Thin filament structural variation contributes to the functional diversity of normal and spaceflight-unloaded muscles.

The purpose of this investigation was to assess muscle fiber size, composition, and in vivo contractile characteristics of the calf muscle of four male crew members during a 17-day spaceflight (SF; Life and Microgravity Sciences Spacelab Shuttle Transport System-78 mission) and eight men during a 17-day bed rest (BR). The protocols and timelines of these two investigations were identical, therefore allowing for direct comparisons between SF and the BR. The subjects' age, height, and weight were 43 ± 2 yr, 183 ± 4 cm, and 86 ± 3 kg for SF and 43 ± 2 yr, 182 ± 3 cm, and 82 ± 4 kg for BR, respectively. Calf muscle strength was examined before SF and BR; on days 2, 8, and12during SF and BR; and on days 2and8of recovery. Muscle biopsies were obtained before and within 3 h after SF (gastrocnemius and soleus) and BR (soleus) before reloading. Maximal isometric calf strength and the force-velocity characteristics were unchanged with SF or BR. Additionally, neither SF nor BR had any effect on fiber composition or fiber size of the calf muscles studied. In summary, no changes in calf muscle strength and morphology were observed after the 17-day SF and BR. Because muscle strength is lost during unloading, both during spaceflight and on the ground, these data suggest that the testing sequence employed during the SF and BR may have served as a resistance training countermeasure to attenuate whole muscle strength loss.

The low intracellular pH and membrane depolarization associated with repeated skeletal muscle stimulation could impair the function of the transverse tubular (t tubule) voltage sensor and result in a decreased sarcoplasmic reticulum Ca2+release and muscle fatigue. We therefore examined the effects of membrane depolarization and low intracellular pH on the t-tubular charge movement. Fibers were voltage clamped in a double Vaseline gap, at holding potential (HP) of −90 or −60 mV, and studied at an internal pH of 7.0 and 6.2. Decreasing intracellular pH did not significantly alter the maximum amount of charge moved, transition voltage, or steepness factor at either HP. Depolarizing HP significantly decreased steepness factor and maximum charge moved and shifted the transition voltage to more positive potentials. Elevated extracellular Ca2+decreased the depolarization-induced reduction in the charge movement. These results indicate that, although the decrease in intracellular pH seen in fatigued muscle does not impair the t-tubular charge movement, the membrane depolarization associated with muscle fatigue may be sufficient to inactivate a significant fraction of the t-tubular charge. However, if t-tubular Ca2+increases, some of the charge may be stabilized in the active state and remain available to initiate sarcoplasmic reticulum Ca2+release.

Spaceflight (SF) has been shown to cause skeletal muscle atrophy; a loss in force and power; and, in the first few weeks, a preferential atrophy of extensors over flexors. The atrophy primarily results from a reduced protein synthesis that is likely triggered by the removal of the antigravity load. Contractile proteins are lost out of proportion to other cellular proteins, and the actin thin filament is lost disproportionately to the myosin thick filament. The decline in contractile protein explains the decrease in force per cross-sectional area, whereas the thin-filament loss may explain the observed postflight increase in the maximal velocity of shortening in the type I and IIa fiber types. Importantly, the microgravity-induced decline in peak power is partially offset by the increased fiber velocity. Muscle velocity is further increased by the microgravity-induced expression of fast-type myosin isozymes in slow fibers (hybrid I/II fibers) and by the increased expression of fast type II fiber types. SF increases the susceptibility of skeletal muscle to damage, with the actual damage elicited during postflight reloading. Evidence in rats indicates that SF increases fatigability and reduces the capacity for fat oxidation in skeletal muscles. Future studies will be required to establish the cellular and molecular mechanisms of the SF-induced muscle atrophy and functional loss and to develop effective exercise countermeasures.

Limb muscles from rats flown in space and after hindlimb unloading (HU) show an increased fatigability, and spaceflight has been shown to result in a reduced ability to oxidize fatty acids. The purpose of this investigation was to determine the effects of HU on the substrate content in fast- and slow-twitch fibers and to assess the substrate utilization patterns in single slow type I fibers isolated from control and HU animals. A second objective was to assess whether HU altered the ability of the heart or limb muscle to oxidize pyruvate or palmitate. After 2 wk of HU, single fibers were isolated from the freeze-dried soleus and gastrocnemius muscles. HU increased the glycogen content in all fiber types, and it increased lactate, ATP, and phosphocreatine in the slow type I fiber. After HU, the type I fiber substrate profile was shifted toward that observed in fast fibers. For example, fiber glycogen increased from 179 ± 16 to 285 ± 25 mmol/kg dry wt, which approached the 308 ± 23 mmol/kg dry wt content observed in the post-HU type IIa fiber. With contractile activity, the type I fiber from the HU animal showed a greater utilization of glycogen and accumulation of lactate compared with the control type I fiber. HU had no effect on the ability of crude homogenate or mitochondria fractions from the soleus or gastrocnemius to oxidize pyruvate or palmitate. The increased fatigability after HU may have resulted from an elevated glycolysis producing an increased cell lactate and a decreased pH.

Soleus muscle fibers were examined electron microscopically from pre- and postflight biopsies of four astronauts orbited for 17 days during the Life and Microgravity Sciences Spacelab Mission (June 1996). Myofilament density and spacing were normalized to a 2.4-μm sarcomere length. Thick filament density (∼1,062 filaments/μm2) and spacing (∼32.5 nm) were unchanged by spaceflight. Preflight thin filament density (2,976/μm2) decreased significantly (P< 0.01) to 2,215/μm2in the overlap A band region as a result of a 17% filament loss and a 9% increase in short filaments. Normal fibers had 13% short thin filaments. The 26% decrease in thin filaments is consistent with preliminary findings of a 14% increase in the myosin-to-actin ratio. Lower thin filament density was calculated to increase thick-to-thin filament spacing in vivo from 17 to 23 nm. Decreased density is postulated to promote earlier cross-bridge detachment and faster contraction velocity. Atrophic fibers may be more susceptible to sarcomere reloading damage, because force per thin filament is estimated to increase by 23%.

Sarcomere disruptions are observed in the adductor longus (AL) muscles following voluntary reloading of spaceflown and hindlimb suspension unloaded (HSU) rat, which resemble lesions in eccentrically challenged muscle. We devised and tested an eccentric contraction (ECCON) test system for the 14‐day HSU rat AL. Six to 7 hours following ECCON, ALs were fixed to allow immunostaining and electron microscopy (EM). Toluidine blue‐stained histology semithin sections were screened for lesion density (#/mm2). Serial semithin sections from the ECCON group were characterized for myosin immunointensity of lesions. Five myofibrillar lesion types were identified in histological semithin sections: focal contractions; wide A‐bands; opaque areas; missing A‐bands; and hyperstretched sarcomeres. Lesion density by type was greater for ECCON than NonECCON ALs (P≤ 0.05; focal contractions and opaque regions). Lesion density (#‐of‐all‐five‐types/mm2) was significantly different (ECCON: 23.91 ± 10.58 vs. NonECCON: 5.48 ± 1.28, P≤ 0.05; ECCON vs. SHAM: 0.00 ± 0.00; P≤ 0.025). PostECCON optimal tension decreased (Poi‐drop, 17.84 ± 4.22%) and was correlated to lesion density (R2= 0.596), but prestretch tension demonstrated the highest correlation with lesion density (R2= 0.994). In lesions, the darkly staining A‐band lost the normally organized thick filament alignment to differing degrees across the different lesion types. Ranking the five lesion types by a measure of lesion length deformation (hypercontracted to hyperstretched) at the light microscopy level, related to the severity of thick filament registry loss across the lesion types at the electron microscopic level. This ranking suggested that the five lesion types seen in semithin sections at the light level represented a lesion progression sequence and paralleled myosin immunostaining loss as the distorted A‐band filaments spread across the hyperlengthening lesion types. Lesion ultrastructure indicated damage involved calcium homeostasis loss (focal contraction lesions) and “thick‐filamentcentering” failure of titin (wide A‐band lesions) in the early stages of lesion development. Anat Rec 254:39–52, 1999. © 1999 Wiley‐Liss, Inc.

Sarcomere disruptions are observed in the adductor longus (AL) muscles following voluntary reloading of spaceflown and hindlimb suspension unloaded (HSU) rat, which resemble lesions in eccentrically challenged muscle. We devised and tested an eccentric contraction (ECCON) test system for the 14-day HSU rat AL. Six to 7 hours following ECCON, ALs were fixed to allow immunostaining and electron microscopy (EM). Toluidine blue-stained histology semithin sections were screened for lesion density (#/mm²). Serial semithin sections from the ECCON group were characterized for myosin immunointensity of lesions. Five myofibrillar lesion types were identified in histological semithin sections: focal contractions; wide A-bands; opaque areas; missing A-bands; and hyperstretched sarcomeres. Lesion density by type was greater for ECCON than NonECCON ALs (P ≤ 0.05; focal contractions and opaque regions). Lesion density (#-of-all-five-types/mm²) was significantly different (ECCON: 23.91 ± 10.58 vs. NonECCON: 5.48 ± 1.28, P ≤ 0.05; ECCON vs. SHAM: 0.00 ± 0.00; P ≤ 0.025). PostECCON optimal tension decreased (Poi-drop, 17.84 ± 4.22%) and was correlated to lesion density (R² = 0.596), but prestretch tension demonstrated the highest correlation with lesion density (R² = 0.994). In lesions, the darkly staining A-band lost the normally organized thick filament alignment to differing degrees across the different lesion types. Ranking the five lesion types by a measure of lesion length deformation (hypercontracted to hyperstretched) at the light microscopy level, related to the severity of thick filament registry loss across the lesion types at the electron microscopic level. This ranking suggested that the five lesion types seen in semithin sections at the light level represented a lesion progression sequence and paralleled myosin immunostaining loss as the distorted A-band filaments spread across the hyperlengthening lesion types. Lesion ultrastructure indicated damage involved calcium homeostasis loss (focal contraction lesions) and “thick-filamentcentering” failure of titin (wide A-band lesions) in the early stages of lesion development. Anat Rec 254:39–52, 1999. © 1999 Wiley-Liss, Inc.

Results from the Russian Cosmos program suggest that the rhesus monkey is an excellent model for studying weightlessness-induced changes in muscle function. Consequently, the purpose of this investigation was to establish the resting levels of selected substrate and enzymes in individual slow- and fast-twitch muscle fibers of the rhesus monkey. A second objective was to determine the effect of an 18-day sit in the Spacelab experiment-support primate facility [Experimental System for the Orbiting Primate (ESOP)]. Muscle biopsies of the soleus and medial gastrocnemius muscles were obtained 1 mo before and immediately after an 18-day ESOP sit. The biopsies were freeze-dried, and individual fibers were isolated and assayed for the substrates glycogen and lactate and for the high-energy phosphates ATP and phosphocreatine. Fiber enzyme activity was also determined for the glycolytic enzymes phosphofructokinase and lactate dehydrogenase (LDH) and for the oxidative markers 3-hydroxyacyl-CoA dehydrogenase (β-OAC) and citrate synthase. Consistent with other species, the fast type II fibers contained higher glycogen content than did the slow type I fibers. The ESOP sit had no significant effects on the metabolic profile of the slow fibers of either muscle or the fast fibers of the soleus. However, the fast gastrocnemius fibers showed a significant decline in phosphocreatine and an increase in lactate. Also, similar to other species, the fast fibers contained significantly higher LDH activities and lower 3-hydroxyacyl-CoA dehydrogenase activities. For the muscle enzymes, the quantitatively most important effect of the ESOP sit occurred with LDH where activities increased in all fiber types postsit except the slow type I fiber of the medial gastrocnemius.

In this study, we determined the contractile properties of single chemically skinned fibers prepared from the medial gastrocnemius (MG) and soleus (Sol) muscles of adult male rhesus monkeys and assessed the effects of the spaceflight living facility known as the experiment support primate facility (ESOP). Muscle biopsies were obtained 4 wk before and immediately after an 18-day ESOP sit, and fiber type was determined by immunohistochemical techniques. The MG slow type I fiber was significantly smaller than the MG type II, Sol type I, and Sol type II fibers. The ESOP sit caused a significant reduction in the diameter of type I and type I/II (hybrid) fibers of Sol and MG type II and hybrid fibers but no shift in fiber type distribution. Single-fiber peak force (mN and kN/m2) was similar between fiber types and was not significantly different from values previously reported for other species. The ESOP sit significantly reduced the force (mN) of Sol type I and MG type II fibers. This decline was entirely explained by the atrophy of these fiber types because the force per cross-sectional area (kN/m2) was not altered. Peak power of Sol and MG fast type II fiber was 5 and 8.5 times that of slow type I fiber, respectively. The ESOP sit reduced peak power by 25 and 18% in Sol type I and MG type II fibers, respectively, and, for the former fiber type, shifted the force-pCa relationship to the right, increasing the Ca2+activation threshold and the free Ca2+concentration, eliciting half-maximal activation. The ESOP sit had no effect on the maximal shortening velocity (Vo) of any fiber type. Voof the hybrid fibers was only slightly higher than that of slow type I fibers. This result supports the hypothesis that in hybrid fibers the slow myosin heavy chain would be expected to have a disproportionately greater influence onVo.

Previously we reported that, after 17‐day bed rest unloading of 8 humans, soleus slow fibers atrophied and exhibited increased velocity of shortening without fast myosin expression. The present ultrastructural study examined fibers from the same muscle biopsies to determine whether decreased myofilament packing density accounted for the observed speeding. Quantitation was by computer‐assisted morphometry of electron micrographs. Filament densities were normalized for sarcomere length, because density depends directly on length. Thick filament density was unchanged by bed rest. Thin filaments/μm2decreased 16–23%. Glycogen filled the I band sites vacated by filaments. The percentage decrease in thin filaments (Y) correlated significantly (P< 0.05) with the percentage increase in velocity (X), (Y = 0.1X + 20%, R2= 0.62). An interpretation is that fewer filaments increases thick to thin filament spacing and causes earlier cross‐bridge detachment and faster cycling. Increased velocity helps maintain power (force × velocity) as atrophy lowers force. Atrophic muscles may be prone to sarcomere reloading damage because force/μm2was near normal, and force per thin filament increased an estimated 30%. © 1998 John Wiley & Sons, Inc. Muscle Nerve 21:1280–1289, 1998.

Previously we reported that, after 17-day bed rest unloading of 8 humans, soleus slow fibers atrophied and exhibited increased velocity of shortening without fast myosin expression. The present ultrastructural study examined fibers from the same muscle biopsies to determine whether decreased myofilament packing density accounted for the observed speeding. Quantitation was by computer-assisted morphometry of electron micrographs. Filament densities were normalized for sarcomere length, because density depends directly on length. Thick filament density was unchanged by bed rest. Thin filaments/μm² decreased 16–23%. Glycogen filled the I band sites vacated by filaments. The percentage decrease in thin filaments (Y) correlated significantly (P &lt; 0.05) with the percentage increase in velocity (X), (Y = 0.1X + 20%, R² = 0.62). An interpretation is that fewer filaments increases thick to thin filament spacing and causes earlier cross-bridge detachment and faster cycling. Increased velocity helps maintain power (force × velocity) as atrophy lowers force. Atrophic muscles may be prone to sarcomere reloading damage because force/μm² was near normal, and force per thin filament increased an estimated 30%. © 1998 John Wiley & Sons, Inc. Muscle Nerve 21:1280–1289, 1998.

Widrick, Jeffrey J., and Robert H. Fitts. Peak force and maximal shortening velocity of soleus fibers after non-weight-bearing and resistance exercise. J. Appl. Physiol.82(1): 189–195, 1997.—This study examined the effectiveness of resistance exercise as a countermeasure to non-weight-bearing-induced alterations in the absolute peak force, normalized peak force (force/fiber cross-sectional area), peak stiffness, and maximal shortening velocity (Vo) of single permeabilized type I soleus muscle fibers. Adult rats were subjected to one of the following treatments: normal weight bearing (WB), non-weight bearing (NWB), or NWB with exercise treatments (NWB+Ex). The hindlimbs of the NWB and NWB+Ex rats were suspended for 14 days via tail harnesses. Four times each day, the NWB+Ex rats were removed from suspension and performed 10 climbs (∼15 cm each) up a steep grid with a 500-g mass (∼1.5 times body mass) attached to their tail harness. NWB was associated with significant reductions in type I fiber diameter, absolute force, normalized force, and stiffness. Exercise treatments during NWB attenuated the decline in fiber diameter and absolute force by almost 60% while maintaining normalized force and stiffness at WB levels. Type I fiberVoincreased by 33% with NWB and remained at this elevated level despite the exercise treatments. We conclude that in comparison to intermittent weight bearing only (J. J. Widrick, J. J. Bangart, M. Karhanek, and R. H. Fitts. J. Appl. Physiol.80: 981–987, 1996), resistance exercise was more effective in attenuating alterations in type I soleus fiber absolute force, normalized force, and stiffness but was less effective in restoring type I fiberVoto WB levels.

Page C86: Fitts, Robert H., and John 0. Holloszy. "Contractile properties of rat soleus muscle: effects of training and fatigue." Pages C88 and C89: Figures and legends corrected.